Method for reducing cadmium accumulation of rice and improving salt tolerance

Abstract

The invention discloses a method for reducing cadmium accumulation of rice and improving salt tolerance. According to the invention, the CDS sequence expression of the OsRBOHA is driven by the OsNAC4 promoter, so that the avoidance capability of the rice to non-uniform stress is remarkably improved, and the stress exposure of a root system is reduced. A soil culture experiment shows that the pOsNAC4: OsRBOHA strain reduces cadmium accumulation of overground parts and grains in non-uniform cadmium polluted soil, and enhances salt tolerance of the overground parts in non-uniform salt stress soil.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to a method for reducing cadmium accumulation and improving salt tolerance in rice. Background Technology

[0002] By 2050, the world's population is projected to reach 9.6 billion, creating an urgent need to increase crop yields and ensure food security to meet the growing global food demand. Rice is a staple food for more than half the world's population, especially in Asia. However, due to global climate change, rapid industrialization, and irrigation methods, soil salinity stress and heavy metal pollution seriously threaten rice yields and food security. Among heavy metals, cadmium (Cd) is highly biotoxic, readily transferring from soil into the food chain and accumulating in edible plant parts, posing a health hazard to humans. Reducing the accumulation of cadmium in rice is essential for reducing cadmium exposure among populations whose staple food is rice.

[0003] As the interface between plants and soil, roots are directly exposed to heavy metal and salt stress in the soil and serve as the first line of defense in plant stress response. Due to the significant spatial variability in the distribution of heavy metals and salts in soil, it remains unclear whether plant roots can sense and avoid areas of high ion stress, thereby reducing root stress exposure. Therefore, enhancing root stress avoidance capabilities using genetic engineering techniques to reduce cadmium accumulation and improve salt tolerance in rice is an important technological goal. Summary of the Invention

[0004] To fill the gap in this technology, this invention uses the NAC domain transcription factor OsNAC4 promoter to link the coding sequence of OsRBOHA, generating the transgenic line pOsNAC4::OsRBOHA, which is applied to reduce cadmium accumulation in rice and improve salt tolerance.

[0005] The objective of this invention can be achieved through the following technical solutions:

[0006] In a first aspect, the present invention protects a recombinant vector containing a CDS of an OsNAC4 promoter and a respiratory burst oxidase homolog OsRBOHA.

[0007] In a specific implementation, the OsNAC4 promoter is shown as SEQ ID NO: 2.

[0008] In a specific implementation, the CDS sequence of the respiratory burst oxidase homolog OsRBOHA is shown in SEQ ID NO: 1.

[0009] By replacing the OsRBOHA promoter with OsNAC4, which is highly sensitive to heavy metal and salt stress, the root system of the pOsNAC4::OsRBOHA strain of this invention exhibits significantly enhanced root avoidance ability against non-uniform heavy metal and salt stress in an agar system under non-uniform heavy metal and salt stress compared to the wild type (Zhonghua 11).

[0010] In a specific implementation plan, the scaffold carrier of the recombinant vector can be the UN1301 carrier.

[0011] Secondly, the present invention protects the transgenic line pOsNAC4::OsRBOHA containing the recombinant vector described above.

[0012] Thirdly, this invention protects the application of the recombinant vector or the transgenic line pOsNAC4::OsRBOHA described above in reducing cadmium accumulation in rice.

[0013] Fourthly, this invention protects the application of the recombinant vector or the transgenic line pOsNAC4::OsRBOHA described above in improving the salt tolerance of rice.

[0014] Fifthly, the present invention protects a method for reducing cadmium accumulation in rice, the method being achieved by introducing the aforementioned recombinant vector into recipient rice.

[0015] Sixthly, the present invention protects a method for improving salt tolerance in rice, the method being achieved by introducing the aforementioned recombinant vector into recipient rice.

[0016] The method described in this invention enhances the root system's ability to avoid non-uniform heavy metal and salt stress, reduces the root system's exposure to ion stress, reduces cadmium accumulation in grains, and improves the salt tolerance of aboveground parts.

[0017] Beneficial effects of the present invention

[0018] 1. This invention, through systematic research, elucidates a method for directional modification of OsRBOHA to enhance root stress avoidance ability, thereby reducing cadmium accumulation in rice grains and improving salt tolerance.

[0019] 2. After targeted modification of OsRBOHA, under hydroponic cadmium stress, the expression level of OsRBOHA induced by cadmium in the pOsNAC4::OsRBOHA line was significantly higher than that in the wild type. Figure 1 ).

[0020] 3. Under hydroponic cadmium stress, the root ROS level of the pOsNAC4::OsRBOHA strain was significantly higher than that of the wild type, and it showed a more sensitive phenotype to cadmium stress. Figure 2 ).

[0021] 4. Under asymmetric cadmium stress agar system, the pOsNAC4::OsRBOHA line responded to cadmium stress more quickly on the cadmium-stressed side, with stronger ROS levels than the wild type. Furthermore, on the safe side, the overall ROS level of the pOsNAC4::OsRBOHA line was also significantly higher than that of the wild type. Six days after unilateral cadmium stress, the number of lateral roots on the asymmetric cadmium-stressed side of the pOsNAC4::OsRBOHA line was significantly higher than that of the wild type, indicating that directional modification of OsRBOHA enhanced systemic ROS transmission under localized cadmium stress, ultimately enhancing root stress avoidance ability. Figure 3 ).

[0022] 5. In the asymmetric cadmium stress soil culture system, the cadmium content in the aboveground parts and grains of the pOsNAC4::OsRBOHA line was significantly lower than that in the wild type, indicating that the directional modification of OsRBOHA reduced cadmium accumulation in the aboveground parts and grains. Figure 4 ).

[0023] 6. In the asymmetric salt stress soil culture system, the aboveground growth and biomass of the pOsNAC4::OsRBOHA line were significantly higher than those of the wild type, indicating that the targeted modification of OsRBOHA enhanced the salt tolerance of the aboveground parts. Figure 5 ).

[0024] 7. Targeted modification of OsRBOHA does not affect the agronomic traits of rice. Figure 6 ). Attached Figure Description

[0025] Figure 1 Information on the construction of pOsNAC4::OsRBOHA and the expression levels of OsRBOHA in wild-type and pOsNAC4::OsRBOHA lines under hydroponic cadmium stress system.

[0026] Figure 2 ROS levels and root elongation in wild-type and pOsNAC4::OsRBOHA strains under hydroponic cadmium stress system.

[0027] Figure 3 The systemic ROS levels and root configurations under asymmetric cadmium stress are shown.

[0028] Figure 4 The cadmium content in the aboveground parts and grains of rice under asymmetric cadmium stress soil culture system.

[0029] Figure 5 The aboveground growth and biomass of rice under asymmetric salt stress soil culture system.

[0030] Figure 6 These are the main agronomic traits of the wild-type and pOsNAC4::OsRBOHA lines in field trials.

[0031] Figure 7 This is a model diagram of a method to reduce cadmium accumulation and improve salt tolerance in rice.

[0032] Figure 8 This is a diagram of the pOsNAC4::OsRBOHA vector. Detailed Implementation

[0033] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0034] Example 1

[0035] The specific implementation process for obtaining the pOsNAC4::OsRBOHA strain is as follows:

[0036] 1) A 2852 bp OsNAC4 promoter was amplified from the DNA of Zhonghua 11 rice, and the CDS of OsRBOHA was amplified from the cDNA of Zhonghua 11. The two fragments were recombined into the UN1301 vector, with EcoRI and SacI restriction sites, to obtain the recombinant plasmid. The recombinant plasmid was transformed into rice by Agrobacterium tumefaciens, and T0 generation lines were obtained. The success of the transgenesis was verified by PCR, and the line was propagated to the T1 generation.

[0037] The following are the primers required for constructing the vector:

[0038] proOsNAC4-F:tatgaccatgattacgaattcAGCTAGCCTGGTGTATATTAAGCTCCA;

[0039] proOsNAC4-R: gattccgtcatCCTTCTCCTCTCGTGAAAATGTTG;

[0040] OsRBOHA-F: aggagaaggATGACGGAATCGAAGGAGTTCG;

[0041] OsRBOHA -R:tctagaggatccccgggtaccTCAGAAATGCTCCTTATGGAATTCG.

[0042] In this embodiment, pOsNAC4::OsRBOHA of two strains of Zhonghua 11 rice were obtained as a background.

[0043] 2) Agrobacterium infection steps:

[0044] After dehulling, the seeds were soaked in 70% alcohol for 2 min and sodium hypochlorite (2%) for 30 min, rinsed several times with sterile water, soaked for 30 min, then blotted dry on sterile filter paper, dried in a laminar flow hood, and placed in induction medium, 12-14 seeds per petri dish. The petri dishes were sealed with sealing film and cultured in a 30℃ light incubator for 4 weeks. Naturally scattered embryogenic callus tissue was picked from the induction medium and subcultured for 1-2 weeks in a 30℃ light incubator. 100 µl of Agrobacterium monoclonal culture was added to 4 ml of YEP (containing 50 mg / L Kana) culture medium and cultured in a shaker for approximately 20-36 h until the OD600 of the culture was measured to be 0.8-1.0. Next, antibiotic screening was performed to induce differentiation and rooting of resistant callus tissue. When the seedlings reached approximately 3 cm in height, they were placed in a rooting medium to promote seedling growth.

[0045] Example 2

[0046] The semi-quantitative detection of OsRBOHA gene expression level in the pOsNAC4::OsRBOHA strain was performed as follows:

[0047] 1) After germinating wild-type and pOsNAC4::OsRBOHA line seeds at 37℃ for 3 days, they were sown on a suspended plastic black net and allowed to grow for 2-3 days.

[0048] 2) Treat the roots in 1 / 2 KimuraB nutrient solution with or without 5 μM CdCl2 for 6 hours, extract RNA, and then reverse transcribe to synthesize cDNA;

[0049] 3) Use the following quantitative PCR primers for PCR amplification to perform semi-quantitative detection of gene transcription:

[0050] qOsRBOHA-F: 5'- GGGAAATGCACAACGAAGTT-3';

[0051] qOsRBOHA-R: 5'- TCTATCCTTGTTTCCCCTCAA-3';

[0052] qOsActin-F: 5'- GACTCTGGTGATGGTGTCAGC-3';

[0053] qOsActin-R: 5'-GGCTGGAAGAGGACCTCAGG-3';

[0054] qOsHistone-F:5'-GGTCAACTTGTTGATTCCCCTCT-3';

[0055] qOsHistone-R: 5'-AACCGCAAAATCCAAAGAACG-3';

[0056] After targeted modification of OsRBOHA, under hydroponic cadmium stress, the expression level of OsRBOHA induced by cadmium in the pOsNAC4::OsRBOHA line was significantly higher than that in the wild type. Figure 1 ).

[0057] Example 3

[0058] ROS levels and root elongation in wild-type and pOsNAC4::OsRBOHA lines.

[0059] 1) After germinating wild-type and pOsNAC4::OsRBOHA line seeds at 37℃ for 3 days, they were sown on a suspended plastic black net and allowed to grow for 2-3 days.

[0060] 2) Treat with 5 μM CdCl2 in 1 / 2 KimuraB nutrient solution for 6 hours with or without adding 5 μM CdCl2, using ROS fluorescent probe (H2DCFDA).

[0061] 3) ROS fluorescent staining was performed on rice roots. Root elongation was measured two days after treatment.

[0062] Under hydroponic cadmium stress, the root ROS level of the pOsNAC4::OsRBOHA line was significantly higher than that of the wild type, and it showed a more sensitive phenotype to cadmium stress. Figure 2 ).

[0063] Example 4

[0064] A split agar system was used for asymmetric stress treatment experiments. First, a 1 / 4 strength Kimura B nutrient solution was prepared and the pH adjusted to 5.7. 1% (w / v) agar powder was added to the nutrient solution, followed by autoclaving at 121°C for 20 minutes. Before the agar medium solidified, heavy metal or salt treatment was added: CdCl2 (0.5–50 μM) or NaCl (10–160 mM). After the agar solidified, agar blocks approximately 7.3 × 2.3 × 1 cm in size were cut using a sterile scalpel and placed side-by-side in petri dishes, leaving a 1 mm gap. In the control group, neither agar block contained the stress treatment; in the asymmetric stress treatment group, only the left agar block contained the specific stress treatment. Two days after germination, the seed roots of rice seedlings were embedded in the 1 mm gap, and the seedlings were allowed to grow for a specific period before phenotypic observation.

[0065] Under asymmetric cadmium stress agar system, the pOsNAC4::OsRBOHA line responded to cadmium stress more quickly on the cadmium-stressed side, exhibiting stronger ROS levels than the wild type. Furthermore, on the safe side, the overall ROS level of the pOsNAC4::OsRBOHA line was also significantly higher than that of the wild type. Six days after unilateral cadmium stress, the number of lateral roots on the asymmetric cadmium-stressed side of the pOsNAC4::OsRBOHA line was significantly higher than that of the wild type, indicating that directional modification of OsRBOHA enhanced systemic ROS transmission under localized cadmium stress, ultimately improving root stress avoidance ability. Figure 3 ).

[0066] Example 5

[0067] Cadmium content in aboveground parts and grains of rice under asymmetric cadmium stress soil culture system.

[0068] 1) The soil that passed through a 2 mm sieve was divided into four portions. One portion was left untreated, two portions were treated with 5 mg / kg Cd, and one portion was treated with 10 mg / kg Cd to maintain a consistent moisture content.

[0069] 2) The first method involves placing four equal portions of untreated soil, referred to as Control; the second method involves placing four equal portions of soil containing 5 mg / kg Cd in a 15*15*15 cm plastic box, referred to as Symmetric Cd; the third method involves placing two equal portions of untreated soil and two equal portions of soil containing 10 mg / kg Cd diagonally, referred to as Asymmetric Cd. Figure 4 As shown, each pot contains 2.5 kg of soil;

[0070] 3) Seven-day-old wild-type pOsNAC4::OsRBOHA strains were placed in... Figure 4 In the apparatus, each material is replicated six times biologically;

[0071] 4) Add water every three days. Weigh the product before adding water to ensure that each treatment has the same moisture content and keep it moist.

[0072] 5) After four weeks of treatment, the root biomass and aboveground cadmium content in the symmetrical and asymmetrical cadmium areas were statistically analyzed.

[0073] Wild-type and pOsNAC4::OsRBOHA strains were cultured until fruit set, and the treatment method was no different from that described above. The containers were changed to 25*25*25cm black containers, and the soil weight was 15kg.

[0074] In the asymmetric cadmium stress soil culture system, the cadmium content in the aboveground parts and grains of the pOsNAC4::OsRBOHA line was significantly lower than that in the wild type, indicating that the directional modification of OsRBOHA reduced cadmium accumulation in the aboveground parts and grains. Figure 4 ).

[0075] Example 6

[0076] The aboveground growth of the pOsNAC4::OsRBOHA line in an asymmetric salt stress soil culture system. The specific implementation process is as follows:

[0077] 1) The soil that passed through a 2 mm sieve was divided into four portions. One portion was left untreated, two portions were treated with 3 g / kg NaCl, and one portion was treated with 6 g / kg NaCl to maintain a consistent moisture content.

[0078] 2) The first method involves placing four equal portions of untreated soil, known as Control. The second method involves placing four equal portions of soil containing 3 g / kg NaCl in a 15*15*15 cm plastic box, known as Symmetric NaCl. The third method involves placing two equal portions of untreated soil and two equal portions of soil containing 6 g / kg NaCl diagonally, known as Asymmetric NaCl. Figure 5 As shown, each pot contains 2.5 kg of soil;

[0079] 3) Seven-day-old wild-type pOsNAC4::OsRBOHA strains were placed in... Figure 5 In the apparatus, each material is replicated six times biologically;

[0080] 4) Add water every three days. Weigh the product before adding water to ensure that each treatment has the same moisture content and keep it moist.

[0081] 5) After four weeks of treatment, the aboveground biomass of symmetrical and asymmetrical salt regions was counted.

[0082] In the asymmetric salt stress soil culture system, the aboveground growth and biomass of the pOsNAC4::OsRBOHA line were significantly higher than those of the wild type, indicating that the directional modification of OsRBOHA enhanced the salt tolerance of the aboveground parts. Figure 5 ).

[0083] Example 7

[0084] Wild-type and pOsNAC4::OsRBOHA lines were planted in Nanjing, and six rice plants from the middle row of each line were taken for agronomical trait analysis. Directional modification of OsRBOHA did not affect rice agronomic traits. Figure 6 ).

[0085] The scope of protection of this invention is not limited to the above embodiments. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in this invention and are protected by the appended claims.

Claims

1. A recombinant vector containing a CDS of an OsNAC4 promoter and a respiratory burst oxidase homolog OsRBOHA.

2. The recombinant vector according to claim 1, characterized in that, The scaffold vector of the recombinant vector is the UN1301 vector.

3. The recombinant vector according to claim 1, characterized in that, The CDS sequence of the respiratory burst oxidase homolog OsRBOHA is shown in SEQ ID NO:

1.

4. The recombinant vector according to claim 1, characterized in that, The OsNAC4 promoter is shown in SEQ ID NO:

2.

5. A transgenic line containing the recombinant vector as described in claim 1 or 2 pOsNAC4::OsRBOHA strains.

6. The application of the recombinant vector according to any one of claims 1-4 or the transgenic line according to claim 5 in reducing cadmium accumulation in rice.

7. The application of the recombinant vector according to any one of claims 1-4 or the transgenic line according to claim 5 in improving the salt tolerance of rice.

8. A method for reducing cadmium accumulation in rice, characterized in that, The method is achieved by introducing the recombinant vector according to any one of claims 1-4 into recipient rice.

9. A method for improving salt tolerance in rice, characterized in that, The method is achieved by introducing the recombinant vector according to any one of claims 1-4 into recipient rice.

Images